CN103748222B - Novel anti-human NGF antibody - Google Patents

Abstract

The present invention provides an anti-human NGF antibody or an antigen-binding fragment thereof that maintains a high neutralizing activity and reduces the risk of side effects such as effects on the fetus and thrombosis, and is excellent in safety, and a human antibody using the antibody or an antigen-binding fragment thereof. NGF is involved in the prophylaxis or treatment of various diseases. An anti-human NGF antibody Fab' fragment, which includes a heavy chain variable region composed of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region composed of the amino acid sequence shown in SEQ ID NO: 4.

Description

Novel anti-human NGF antibody

technical field

The present invention relates to novel anti-human NGF antibodies, more specifically to Fab' fragments of anti-human NGF antibodies.

Background technique

Nerve growth factor (NGF) is one of the humoral factors collectively called neurotrophic factors, which play an important role in the occurrence, differentiation and function maintenance of neurons in organisms. As receptors for NGF, there are known high-affinity trkA receptors (receptor-type tyrosine kinases) and low-affinity p75NTR receptors. Among them, it has been reported that p75NTR binds to all neurotrophic factors and is involved in apoptosis in the process of neurogenesis, but its role has not been fully clarified. On the other hand, it is known that NGF and trkA receptor knockout mice show the same phenotype (Non-Patent Document 1), and it is considered that the physiological action of NGF is mainly expressed through the trkA receptor.

In 1993, it was reported that pain could be induced by administering NGF to rats (Non-Patent Document 2), and later it was reported that systemic muscle pain could be induced by intravenous administration of NGF to humans, and by local administration, except In addition to systemic effects, it induces hyperalgesia and allodynia at the injection site (Non-Patent Document 3). In addition, it has been reported that trkA receptor knockout mice lack pain sensation (Non-Patent Document 4), and NGF is considered to be a molecule closely related to the expression of pain. Regarding the correlation with the pathological state of human pain, it has been confirmed that the expression of NGF/trkA in the articular cartilage of degenerative joint disease (osteoarthritis; OA) is increased (Non-Patent Document 6), rheumatoid arthritis (Non-Patent Document 7) , NGF levels increased in patients with interstitial cystitis (Non-Patent Document 8).

From these facts, it is known that if a monoclonal antibody that specifically binds to NGF and has an activity to inhibit its action can be developed, it can be expected to be useful for the treatment, prevention, and diagnosis of various diseases related to NGF represented by pain .

So far, humanized anti-human NGF antibodies Tanezumab (Patent Document 1) and PG110 (Patent Document 2) as humanized anti-human NGF antibodies, and REGN475 as a fully human anti-human NGF antibody (Patent Document 3), Fulranumab (Patent Document 4), and MEDI-578 (Patent Document 5). Among them, tanezumab was the first to be developed, and it has been reported from its clinical results that it has shown a positive effect on joint pain associated with degenerative joint disease, chronic low back pain, and bladder pain associated with interstitial cystitis. Strong and broad analgesic effect (Non-Patent Documents 9 to 11).

In general, as the main elements for specifying the effective dosage of an antibody drug, the neutralizing activity of the antibody against the antigen, the amount of the antigen present in the body, and the improvement of the neutralizing activity correspond to the reduction of the dosage. It can be said that the result is an extremely beneficial improvement related to the economic burden of patients and the reduction of medical costs. In addition, subcutaneous administration is also possible as long as the dose can be reduced. With regard to subcutaneous administration, if certain conditions are met, it has the greatest advantage of being able to perform self-injection at home. In addition, in general, intravenous administration requires a certain period of time to implement drip administration in most cases. In contrast, subcutaneous administration Drugs are also useful in that they can be administered as pills, and it is desirable for both physicians and patients to be able to choose between intravenous and subcutaneous administration preparations. However, in general, the volume that can be administered in one subcutaneous administration is about 1 mL, which is a small amount. In order to express the drug effect, it is necessary to contain sufficient antibody in this liquid volume. In addition, unlike intravenous administration, bioavailability must also be considered. That is, in order to realize preparations for subcutaneous administration, it is required to produce antibodies that are excellent in solubility and exhibit sufficient drug efficacy even at low dosages. Therefore, the acquisition of antibodies with higher neutralizing activity against NGF than existing antibodies is useful for the treatment of NGF-related diseases and the improvement of convenience thereof.

In addition, as mentioned above, NGF is an important factor for the development of neurons, and the development of drugs that inhibit the function of NGF requires sufficient research from the viewpoint of safety. In particular, as one of the matters that should be studied in terms of safety, the influence on the fetus can be mentioned. Up to now, regarding the functional inhibition of NGF, it has been reported that NGF mutation is the cause of congenital analgesia (non-patent document 5); in animal experiments, if pregnant guinea pigs are made to produce autoantibodies against NGF to inhibit , the newborn guinea pigs produced showed painless symptoms (non-patent literature 12). In addition, experiments using NGF and trkA-deficient mice have also confirmed that the development of embryonic sensory and sympathetic neurons is inhibited due to the absence of NGF action (Non-Patent Documents 4 and 13). From these facts, it can be understood that NGF Is an essential factor for the occurrence of early neurodevelopment. On the other hand, NGF-related diseases also include: interstitial cystitis (more than half of the patients are under 44 years old, and 90% of the patients are women (Non-Patent Document 14)), chronic low back pain (average age is 40 to 50 years old, more than 50% of patients are women (Non-Patent Document 15-17)), migraine (mostly in 15-40 years old, 80% of patients are women (Non-Patent Document 18)) and other women in the right age for pregnancy Diseases with moderate to high rates of occurrence. From this situation, it can be seen that it is extremely important to avoid the risk of side effects on the fetus of a pregnant woman when developing an anti-NGF antibody as a drug.

In addition, another risk factor in the case of developing an anti-NGF antibody as a drug is the formation of an immune complex (IC). The immune complex formed by the combination of antibody and antigen is usually disposed of in the reticuloendothelial system of the spleen, liver, etc., but in pathological conditions such as immune abnormalities, when the size of the formed IC is large, the IC loses solubility, and the thrombus forms The risk is increased and it accumulates in the glomeruli, which is associated with the development of nephritis. IgG is a bivalent antibody, and when the antigen is polyvalent, IC can obtain various sizes due to lattice formation. The size of the IC depends on the amount and ratio of the antibody to the antigen, the affinity of the antibody, etc. For example, it has been reported that the anti-VEGF antibody Bevacizumab (trade name: Abastin) is an IgG1 antibody that binds to dimer VEGF to form an IC , can induce thrombosis. Specifically, when abastin and VEGF were administered to human FcγRIIα receptor Tg mice, formation of pulmonary artery thrombus was observed (Non-Patent Document 19). It has also been reported that patients with metastatic cancer who received chemotherapy and Abastine had a higher incidence of arterial thrombosis compared with a placebo group receiving chemotherapy alone (Non-Patent Document 20). NGF also plays a physiological role by forming a dimer in vivo. Therefore, in the development of anti-NGF antibody drugs, it is desired to avoid the risk of IC formation and further improve safety.

Thus, it can be seen that the acquisition of anti-NGF antibodies with excellent safety, which maintains high neutralizing activity and reduces the risk of side effects such as the impact on the fetus and thrombosis, is very important for the treatment or prevention of various diseases related to NGF. extremely important.

prior art literature

patent documents

Patent Document 1: WO2004/058184

Patent Document 2: WO2005/061540

Patent Document 3: WO2009/023540

Patent Document 4: WO2005/019266

Patent Document 5: WO2006/077441

non-patent literature

Non-Patent Document 1: Conover JC, et al, Rev Neurosci.1997, 8:13-27.

Non-Patent Document 2: Lewin GR, et al, J Neurosci.1993, 13:2136-48.

Non-Patent Document 3: Petty BG, et al, Ann Neurol.1994, 36:244-6.

Non-Patent Document 4: Smeyne RJ, et al, Nature.1994,368:246-9.

Non-Patent Document 5: Indo Y, et al, Nat Genet.1996, 13:485-8.

Non-Patent Document 6: Iannone F, et al, Rheumatology 2002, 41:1413-8.

Non-Patent Document 7: Aloe L, et al, Clin Exp Rheumatol.1997, 15:433-8.

Non-Patent Document 8: Lowe EM, et al, Br J Urol.1997, 79:572-7.

Non-Patent Document 9: Lane NE, et al, N Engl J Med.2010,363:1521-31.

Non-Patent Document 10: Evans RJ, et al, J Urol.2011,185:1716-21.

Non-Patent Document 11: Katz N, et al, Pain.2011, in press

Non-Patent Document 12: Johnson EM Jr, et al, Science.1980, 210:916-8.

Non-Patent Document 13: Crowley C, et al, Cell.1994, 76:1001-11.

Non-Patent Document 14: Payne CK, et al, J Urol.2007,177:2042-9.

Non-Patent Document 15: Manchikanti L, et al, Pain Physician.2010, 13:E279-92.

Non-Patent Document 16: Wilkens P, et al, JAMA.2010, 304:45-52.

Non-Patent Document 17: Buynak R, et al, Expert Opin Pharmacother.2010, 11:1787-804.

Non-Patent Document 18: Sakai F, et al, Cephalalgia.1997, 17:15-22.

Non-Patent Document 19: Meyer T, et al, J Thromb Haemost.2009, 7:171-81.

Non-Patent Document 20: Scappaticci FA, et al, J Natl Cancer Inst.2007,99:1232-9.

Contents of the invention

The problem to be solved by the invention

The object of the present invention is to provide an anti-human NGF antibody or an antigen-binding fragment thereof that maintains high neutralizing activity and reduces the risk of side effects such as effects on the fetus and thrombosis and is excellent in safety.

method used to solve the problem

The present invention includes the following inventions as medically or industrially useful substances and methods.

[1] An anti-human NGF antibody Fab' fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4.

[2] The Fab' fragment according to [1], wherein the heavy chain constant region of the Fab' fragment is a human Igγ1 constant region.

[3] The Fab' fragment according to [1], wherein the light chain constant region of the Fab' fragment is a human Igκ constant region.

[4] The Fab' fragment according to [1], wherein the heavy chain constant region of the Fab' fragment is a human Igγ1 constant region, and the light chain constant region of the Fab' fragment is a human Igκ constant region.

[5] The Fab' fragment as described in [1], which comprises a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 16 and a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 12 light chain.

[6] The Fab' fragment according to any one of [1] to [5], which is conjugated with polyethylene glycol.

[7] A polynucleotide comprising a sequence encoding the heavy chain fragment of the Fab' fragment according to any one of [1] to [6].

[8] A polynucleotide comprising a sequence encoding the light chain of the Fab' fragment according to any one of [1] to [6].

[9] An expression vector comprising the polynucleotide described in [7] and/or [8].

[10] A host cell transformed with the expression vector of [9].

[11] The host cell according to [10], which is selected from the group consisting of the following (a) and (b),

(a) Using a polynucleotide comprising a sequence encoding the heavy chain fragment of the Fab' fragment described in any one of [1] to [6] and a polynucleotide comprising a sequence encoding the light chain of the Fab' fragment The host cell transformed with the expression vector; and

(b) Using an expression vector comprising a polynucleotide comprising the sequence encoding the heavy chain fragment of the Fab' fragment described in any one of [1] to [6] and comprising a sequence comprising the light chain encoding the Fab' fragment The host cell is transformed with the polynucleotide expression vector.

[12] A method for producing the Fab' fragment described in any one of [1] to [6], which comprises culturing the host cell described in [10] or [11] to produce an anti-human NGF antibody Fab' fragment expressive steps.

[13] A drug for treating pain comprising the Fab' fragment according to any one of [1] to [6].

[14] The therapeutic agent according to [13], wherein the pain is joint pain accompanied by degenerative joint disease.

[15] A method for preventing or treating pain, comprising the step of administering the Fab' fragment according to any one of [1] to [6].

[16] The method according to [15], wherein the pain is joint pain accompanied by degenerative joint disease.

[17] The Fab' fragment according to any one of [1] to [6], which is used for the prevention or treatment of pain.

[18] The Fab' fragment according to [17], wherein the pain is joint pain accompanied by degenerative joint disease.

Invention effect

The anti-human NGF antibody Fab' fragment of the present invention is useful for the prevention or treatment of various diseases in which human NGF is involved in the pathogenesis. Due to its high neutralizing activity, the Fab' fragment of the anti-human NGF antibody of the present invention brings advantages in clinical applications such as reduction of dosage, expansion of administration interval, improvement of administration method (such as subcutaneous injection), etc. Excellent improvement, and the impact on the fetus, the risk of side effects such as thrombosis are reduced, the safety is very good, and it has a significant contribution to the prevention and treatment of various diseases in which human NGF participates in the pathogenesis.

Description of drawings

FIG. 1 shows the time-dependent changes in antibody retention in the plantar of a mouse collagen-induced arthritis model.

Detailed ways

The present invention will be described in detail below.

The inventors of the present invention have repeatedly conducted quite original research on the preparation of anti-human NGF antibodies or antigen-binding fragments thereof, and as a result, successfully produced anti-human NGF antibodies that maintain high neutralizing activity and reduce the risk of side effects such as effects on the fetus and thrombosis. An anti-human NGF antibody Fab' fragment with excellent safety.

The basic structure of antibody molecules is common among various types, and consists of a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with a molecular weight of 20,000 to 30,000. Heavy chains are usually composed of polypeptide chains containing about 440 amino acids, each of which has a characteristic structure, corresponding to IgG, IgM, IgA, IgD, and IgE called γ, μ, α, δ, ε chains. Furthermore, IgG includes IgG1, IgG2, IgG3, and IgG4, which are called γ1, γ2, γ3, and γ4, respectively. Light chains generally consist of polypeptide chains comprising about 220 amino acids, and two types, L-type and K-type, are known and are called λ and κ chains, respectively. Regarding the peptide composition of the basic structure of an antibody molecule, two homologous heavy chains and two homologous light chains are bonded by disulfide bonds (S-S bonds) and non-covalent bonds, and the molecular weight is 150,000 to 190,000 . Both light chains can be paired with either heavy chain. Individual antibody molecules are often formed from the same two light chains and the same two heavy chains.

There are four SS bonds in the heavy chain (five in the μ and ε chains) and two in the light chain, forming a ring every 100-110 amino acid residues, and the three-dimensional structure is similar between the rings , are called structural units or domains. For the domains located at the N-terminus of both the heavy chain and the light chain, even if it is a standard product from the same class (subclass) of the same animal, its amino acid sequence is not constant, and it is called a variable region. The domains are referred to as the heavy chain variable region ( _VH ) and the light chain variable region ( _VL ), respectively. Thus, the amino acid sequence toward the C-terminal side is basically constant in each class or subclass, and is called a constant region, and each domain is represented by _CH 1 , CH ² , CH ³ , or _CL .

The epitope of an antibody is composed of _VH and _VL , and the specificity of binding is determined by the amino acid sequence of the site. On the other hand, biological activities such as binding to complement or various cells reflect differences in the constant region structures of various Igs. The variability of the variable regions of the heavy and light chains is roughly limited to three small hypervariable regions present in both chains, these regions are called complementarity determining regions (CDRs; CDR1, CDR2, respectively, from the N-terminal side , CDR3). The remainder of the variable region, called the framework region (FR), is more constant.

The region between the _CH1 domain and the _CH2 domain of the heavy chain constant region of an antibody is called the hinge region, which contains a large number of proline residues and contains multiple interchains that connect the two heavy chains. SS key. For example, each hinge region of human IgG1, IgG2, IgG3, and IgG4 contains 2, 4, 11, and 2 cysteine residues constituting SS bonds between heavy chains, respectively. The hinge region is a region highly sensitive to proteolytic enzymes such as papain and pepsin. When the antibody is digested with papain, the heavy chain is cut at a position closer to the N-terminal side than the SS bond between the heavy chains in the hinge region, and is decomposed into two Fab fragments and one Fc fragment. The Fab fragment consists of a light chain and a heavy chain fragment comprising the heavy chain variable region ( _VH ), _CHI domain and part of the hinge region. When the antibody is digested with pepsin, the heavy chain is cleaved at a position closer to the C-terminal side than the SS bond between the heavy chains in the hinge region to generate an F(ab') ₂ fragment. The F(ab') ₂ fragment is a fragment of a dimeric structure in which two Fab' fragments are bonded via an inter-heavy-chain SS bond in the hinge region. The Fab' fragment consists of a light chain and a heavy chain fragment comprising the heavy chain variable region (V _H ), the _CH1 domain, and a portion of the hinge region containing the half SS bonds that form the inter-heavy chains. Cystine residues. Fab fragments, F(ab') ₂ fragments, and Fab' fragments all contain variable regions and have antigen-binding activity.

The Fab' fragment of the anti-human NGF antibody of the present invention successfully produced by the present inventors is a Fab' fragment having the following characteristics.

An anti-human NGF antibody Fab' fragment, which includes a heavy chain variable region composed of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region composed of the amino acid sequence shown in SEQ ID NO: 4.

Specifically, the present inventors used human monoclonal antibody development technology "VelocImmune" (VelocImmune antibody technology; Regeneron Corporation (US Patent No. 6,596,541)) to produce antibodies by using antibodies from various biological activity tests and physical property tests Through screening, the Fab' fragment of the anti-human NGF antibody of the present invention was successfully identified. For the Velocimmune technique, transgenic mice in which endogenous immunoglobulin heavy and light chain variable regions are replaced with the corresponding human variable regions are immunized with the antigen of interest (e.g., human βNGF), and then, Lymphoid cells from mice expressing antibodies are obtained, and hybridomas are produced by cell fusion with mouse myeloma cells. Next, the hybridoma cells are screened for the production of antibodies that specifically bind to the target antigen and have the desired neutralizing activity. Here, the antibody produced is an antibody having a variable region of a human antibody and a constant region of a mouse antibody (also referred to as a chimeric antibody in this specification). Next, when an antibody that specifically binds to the target antigen and has the desired neutralizing activity is identified, the DNA encoding the variable region of the heavy chain and the light chain of the antibody is isolated from the hybridoma cell, and the This DNA is ligated to DNA encoding the constant regions of the heavy and light chains of human antibodies of the desired class. The thus obtained genes encoding the heavy chain and light chain are expressed in cells (for example, CHO cells) to produce antibody molecules. The heavy and light chains of antibodies produced by this method are those of "fully human" antibodies derived from human immunoglobulin genes.

For those skilled in the art, the Fab' fragment of the anti-human NGF antibody of the present invention can be based on the sequence information of its heavy chain variable region and light chain variable region disclosed in the specification of this application, and can be easily obtained by using methods known in the art. ground production. Preferably, the anti-human NGF antibody Fab' fragment of the present invention can be obtained by combining its heavy chain variable region and light chain variable region with a part of the heavy chain constant region (including _CH1 domain, and containing A part of the hinge region with cysteine in the hinge region) and the constant region of the light chain are connected to make a Fab' fragment of a fully human antibody. Specifically, a heavy chain variable region gene fragment having a nucleotide sequence encoding the heavy chain variable region amino acid (SEQ ID NO: 6) of the Fab' fragment of the present invention and a light chain encoding the Fab' fragment of the present invention were produced. The light chain variable region gene fragment of the base sequence of the variable region amino acid (SEQ ID NO: 4). Then, genes for the Fab' fragment of a fully human antibody are produced by linking the variable region genes of the heavy chain and light chain to the human antibody heavy chain constant region and light chain constant region genes of an appropriate class. Next, the gene is ligated into an appropriate expression vector and introduced into cultured cells. Finally, the cultured cells are cultured so that monoclonal Fab' fragments can be obtained from the culture supernatant.

A gene fragment encoding the amino acids of the heavy chain variable region and the light chain variable region of the Fab' fragment of the present invention, for example, can be based on a base sequence designed based on the amino acid sequences of the heavy chain variable region and the light chain variable region , synthesized using a gene synthesis method known in the art. As such a gene synthesis method, various methods known to those skilled in the art, such as the antibody gene synthesis method described in WO90/07861, can be used.

Next, the above-mentioned variable region gene fragments are ligated to the constant region genes of human antibodies to prepare fully human antibody Fab' fragment genes. The constant region of a human antibody can be selected from any subclass of constant region (for example, as the constant region of γ1, γ2, γ3 or γ4 of the heavy chain, as the constant region of the λ or κ chain of the light chain), but as the constant region of the heavy chain , human Igγ1 can be preferably used, and as the light chain constant region, human Igκ can be preferably used.

After producing the fully human antibody Fab' fragment gene, introduction of the gene into an expression vector, introduction of the expression vector into cultured cells, cultivation of cultured cells, and production of the Fab' fragment can be performed by various methods known in the art. Purification etc.

Examples of expression vectors linked to the antibody genes obtained as described above include GS vectors pEE6.4 and pEE12.4 (Lonza Biologics), and there are no particular limitations as long as the antibody genes can be expressed. In addition, expression vectors having human Ig constant region genes in advance, such as AG-γ1 and AG-κ (for example, see WO94/20632), can also be used.

The above-mentioned expression vectors are introduced into cultured cells by, for example, the calcium phosphate method, electroporation and the like.

As cultured cells into which expression vectors are introduced, cultured cells such as CHO-K1SV cells, CHO-DG44 cells, and 293 cells can be used, and cultured by conventional methods.

After the above culture, the Fab' fragments accumulated in the culture supernatant can be purified by various column chromatography, for example, column chromatography using KappaSelect or the like can be used.

The Fab' fragment of the present invention can be produced by the aforementioned recombinant expression method, but it is also possible to produce a full-length antibody first, then digest it with pepsin, and reduce the obtained F(ab')2 fragment with 2-mercaptoethanol, etc. Reagents are processed to make Fab' fragments.

The anti-human NGF antibody Fab' fragment of the present invention can be easily obtained preferably by synthesizing a DNA comprising a base sequence encoding the heavy chain variable region amino acid sequence shown in SEQ ID NO: 6 using a method known in the art , and a DNA comprising a base sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO: 4, which are connected to an appropriate type of human antibody constant region gene, preferably a human Igγ1 constant region gene for the heavy chain, and for the heavy chain For the light chain, the human Igκ constant region gene is preferred, and a fully human antibody Fab' fragment gene is constructed, and the gene is introduced into an expression vector using various methods known in the art, and the expression vector is introduced into cultured cells, and the cultured cells are subjected to Cultivation and purification of Fab' fragments from the resulting culture. The DNA comprising the nucleotide sequence encoding the heavy chain variable region amino acid sequence shown in SEQ ID NO:6 preferably includes the nucleotide sequence shown in SEQ ID NO:5. The DNA comprising the nucleotide sequence encoding the light chain variable region amino acid sequence shown in SEQ ID NO: 4 preferably includes the nucleotide sequence shown in SEQ ID NO: 3.

In this specification, a "Fab'fragment" is a monovalent antibody fragment composed of a light chain and a heavy chain fragment including a heavy chain variable region (V _H ), a _CH1 domain, and a part of the hinge region. Some of them contain at least one cysteine residue other than the cysteine residue constituting the SS bond between the heavy chain and the light chain (also referred to as "hinge region cysteine" in this specification). The cysteine in the hinge region can be used as a modification site for polyethylene glycol described later. The number of cysteines in the hinge region in the Fab' fragment can vary from one to several depending on the type of antibody used, and can be easily adjusted by those skilled in the art. For example, when making Fab' fragments of the human IgG1 class (usually with 2 hinge cysteines in the hinge region), in the hinge region of the heavy chain, at the coding site for the initial hinge cysteine A stop codon is inserted between the coding site of the cysteine in the second hinge region, so that a Fab' fragment with one cysteine in the hinge region can be produced, and a cysteine in the second hinge region can be produced. By inserting a stop codon after the coding site for amino acid, a Fab' fragment having two hinge cysteines in the hinge region can be produced.

The heavy chain fragment of the preferred anti-human NGF antibody Fab' fragment of the present invention comprising the heavy chain variable region and the human Igγ1 constant region part of the amino acid sequence shown in SEQ ID NO: 6 is represented by SEQ ID NO: 10, SEQ ID NO: 14. Or a heavy chain fragment composed of the amino acid sequence shown in SEQ ID NO: 16. DNA comprising the base sequence encoding the heavy chain fragment of the anti-human NGF antibody Fab' fragment composed of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 16, preferably SEQ ID NO: 9, SEQ ID NO: 13, Or the base sequence shown in SEQ ID NO: 15. The light chain of the preferred anti-human NGF antibody Fab' fragment of the present invention comprising the light chain variable region composed of the amino acid sequence shown in SEQ ID NO: 4 and the human Igκ constant region is composed of the amino acid sequence shown in SEQ ID NO: 12 light chain. The DNA comprising the nucleotide sequence encoding the light chain of the anti-human NGF antibody Fab' fragment composed of the amino acid sequence shown in SEQ ID NO: 12 preferably includes the nucleotide sequence shown in SEQ ID NO: 11.

Examples of the preferred anti-human NGF antibody Fab' fragment of the present invention comprising a heavy chain fragment composed of the amino acid sequence represented by SEQ ID NO: 10 and a light chain composed of the amino acid sequence represented by SEQ ID NO: 12 include the following: Fully human 1-15 (N52D) antibody Fab' fragment described in the Examples. Examples of the preferred anti-human NGF antibody Fab' fragment of the present invention comprising a heavy chain fragment composed of the amino acid sequence represented by SEQ ID NO: 14 and a light chain composed of the amino acid sequence represented by SEQ ID NO: 12 include the following: The Fab' fragment of the fully human 1-15 (N52D-A) antibody described in the Examples. Examples of the preferred anti-human NGF antibody Fab' fragment of the present invention comprising a heavy chain fragment composed of the amino acid sequence represented by SEQ ID NO: 16 and a light chain composed of the amino acid sequence represented by SEQ ID NO: 12 include the following: The Fab' fragment of the fully human 1-15 (N52D-P) antibody described in the Examples.

The present invention also includes the following anti-human NGF antibody Fab' fragment, said anti-human NGF antibody Fab' fragment includes: comprising CDR1 composed of the 31st to 35th amino acid sequence of SEQ ID NO: 6, consisting of the 50th to 35th amino acid sequence of SEQ ID NO: 6 CDR2 composed of the 65th amino acid sequence, and the heavy chain variable region of CDR3 composed of the 98th to 110th amino acid sequence of SEQ ID NO: 6, and CDR1 composed of the 24th to 39th amino acid sequence of SEQ ID NO: 4, consisting of The light chain variable region of CDR2 composed of the 55th to 61st amino acid sequence of SEQ ID NO: 4, and the CDR3 composed of the 94th to 102nd amino acid sequence of SEQ ID NO: 4. In addition, those skilled in the art can also easily prepare such Fab' fragments according to the aforementioned procedures.

The Fab' fragment of the anti-human NGF antibody of the present invention can also be modified by bonding polyethylene glycol (PEG) to cysteine in its hinge region. Bonding of PEG to Fab' fragments can be performed using methods known in the art (eg European Patent EP0948544). In the present invention, PEG or its derivatives having any average molecular weight of linear or branched chains can be used, and those skilled in the art can easily select according to the purpose of use. For example, in tumor tissues and inflammatory reactions, the permeability of blood vessels is significantly increased compared with normal tissues, and the substances that arrive tend to leak out of blood vessels and accumulate in tumors and inflammatory tissues (EPR effect). It is also known that substances with a small molecular weight are easily reabsorbed by blood vessels, whereas substances with a large molecular weight are difficult to be reabsorbed. Therefore, in order to improve the retention of the Fab' fragment in the lesion tissue, it can be bonded with PEG with a high average molecular weight (for example, about 40000Da), and it can be combined with a low average molecular weight (for example, about 10000Da) of PEG for bonding. In addition, in order to facilitate the bonding of PEG and cysteine in the hinge region, a derivative of PEG can be used. For example, a PEG derivative to which a thiol reactive group such as maleimide is bonded can be used as described in the examples below to allow the thiol group of the cysteine in the hinge region to react with the maleimide group. covalently bonded. Generally, the average molecular weight of PEG is about 500 Da to about 50000 Da, preferably about 5000 Da to about 40000 Da, more preferably about 10000 Da to about 40000 Da.

The anti-human NGF antibody Fab' fragment of the present invention binds to human NGF. Methods for measuring the binding activity of the obtained anti-human NGF antibody Fab' fragment to human NGF include methods such as ELISA and FACS. For example, in the case of using ELISA, human βNGF is immobilized on an ELISA plate, Fab' fragments are added to it and reacted, and then a secondary antibody such as an anti-kappa antibody labeled with an enzyme such as horseradish peroxidase (HRP) is used. After the reaction and washing, the binding of the secondary antibody is identified by an activity measurement using a reagent for detecting its activity (for example, a TMB chromogenic reagent in the case of HRP labeling), or the like. In addition, the anti-human NGF antibody Fab' fragment of the present invention also includes Fab' fragments that bind to NGF derived from other animals (such as mouse NGF) in addition to human NGF, and the binding activity to these proteins can also be measured.

The anti-human NGF antibody Fab' fragment of the present invention has neutralizing activity to human NGF. When used in this specification, the "neutralizing activity" of the Fab' fragment of an anti-human NGF antibody refers to the activity of inhibiting any biological activity brought about by NGF by binding to NGF, one or more of NGF can be Biological activity is used as an index to evaluate. Such neutralizing activity includes, for example, binding inhibitory activity between NGF and trkA as its receptor, NGF-trkA signal-mediated intracellular calcium influx inhibitory activity, and NGF-dependent cell survival signal inhibitory activity. Evaluation was performed using the method described in Examples described later.

In order to evaluate the effect of the anti-human NGF antibody Fab' fragment of the present invention in more detail, an in vivo test can be used. For example, the in vivo drug efficacy of the Fab' fragment can be evaluated using an analgesic effect test using a mouse arthritis model, etc. as described in Examples described later, and the focal tissue retention effect can also be evaluated using a lesion migration test.

In addition, risk assessment of side effects can also be performed on the anti-human NGF antibody Fab' fragment of the present invention. For example, the possibility of the Fab' fragment of the anti-human NGF antibody of the present invention affecting the fetus can be evaluated by using a placenta passage test after administration of the Fab' fragment in a pregnant animal as described in the Examples below. In addition, the size of the IC formed between the anti-human NGF antibody Fab' fragment of the present invention and NGF can be measured using the immune complex (IC) formation test with NGF as described in the Examples below, and the effect on the induction of thrombus formation can be measured. possibility to evaluate.

In addition, methods for evaluating various stability (such as thermal stability, long-term storage stability, and high-concentration stability) of the anti-human NGF antibody Fab' fragment of the present invention include, for example, measurement by differential scanning calorimetry or storage Methods for the determination of aggregate formation in .

The Fab' fragment of the anti-human NGF antibody of the present invention can be purified according to need, prepared by a conventional method, and applied to joint pain associated with degenerative joint disease (OA pain), arthralgia associated with rheumatism, and cancer pain , neuropathic pain, chronic low back pain, postoperative pain, postherpetic neuralgia, painful diabetic neuropathy, fracture pain, bladder pain syndrome and other pain, interstitial cystitis, acute pancreatitis, chronic Treatment of diseases in which NGF is involved, such as pancreatitis and endometriosis.

The Fab' fragment of the anti-human NGF antibody of the present invention can be preferably used as a therapeutic agent for pain, more preferably as a therapeutic agent for joint pain associated with degenerative joint disease. Examples of dosage forms of these therapeutic agents include parenteral preparations such as injections and drips, and are preferably administered by intravenous administration, subcutaneous administration, or the like. In addition, when formulating, carriers and additives corresponding to these dosage forms can be used within a pharmaceutically acceptable range.

In the above formulation, the amount of the anti-human NGF antibody Fab' fragment of the present invention to be added varies depending on the degree of the patient's symptoms, age, dosage form of the preparation used, or the binding potency of the antibody. For example, 0.001 mg/ kg to about 100mg/kg.

The present invention also provides a polynucleotide comprising the sequence encoding the Fab' fragment of the anti-human NGF antibody of the present invention and an expression vector comprising the polynucleotide. The present invention also provides a polynucleotide comprising the sequence encoding the heavy chain variable region of the anti-human NGF antibody Fab' fragment of the present invention, and a sequence comprising the sequence encoding the light chain variable region of the anti-human NGF antibody Fab' fragment of the present invention polynucleotides, and expression vectors comprising any one or both of them. As long as the expression vector of the present invention can express the gene encoding the Fab' fragment of the present invention or its heavy chain variable region and/or light chain variable region in various host cells of prokaryotic cells and/or eukaryotic cells, and The vectors for producing these polypeptides are not particularly limited. Examples thereof include plasmid vectors, viral vectors (eg, adenovirus, retrovirus), and the like. The expression vector of the present invention preferably comprises a polynucleotide comprising a sequence encoding the heavy chain fragment or light chain of the aforementioned Fab' fragment of the present invention, or a polynucleotide comprising a sequence encoding the heavy chain fragment of the Fab' fragment of the present invention , and a polynucleotide comprising a sequence encoding the light chain of a Fab' fragment of the invention.

The expression vector of the present invention may contain a gene encoding the Fab' fragment of the anti-human NGF antibody of the present invention, or a gene encoding the heavy chain variable region and/or the variable region of the light chain of the Fab' fragment of the anti-human NGF antibody of the present invention, and a promoter that can be functionally linked to the gene. As a promoter for expressing the gene encoding the Fab' fragment of the present invention or encoding its heavy chain variable region and/or light chain variable region in bacteria, when the host is a bacterium of the genus Escherichia, it can be Examples include Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like. Examples of promoters for expression in yeast include PH05 promoter, PGK promoter, GAP promoter, and ADH promoter, and examples of promoters for expression in Bacillus bacteria include SL01 promoter, SP02 promoter, penP promoter etc. In addition, when the host is a eukaryotic cell such as a mammalian cell, a promoter derived from SV40, a retrovirus promoter, a heat shock promoter, and the like can be mentioned.

When bacteria, especially Escherichia coli are used as host cells, the expression vector of the present invention may further contain a start codon, a stop codon, a terminator region and a replicable unit. On the other hand, when yeast, animal cells, or insect cells are used as hosts, the expression vector of the present invention may contain start codons and stop codons. In addition, in this case, an enhancer sequence, an untranslated region on the 5' side and a 3' side of a gene encoding the Fab' fragment of the present invention or its heavy chain variable region or light chain variable region, and a secretion signal may be included. sequence, splice site, polyadenylation site or replicable unit, etc. In addition, generally used selection markers (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) may be contained according to the purpose.

The present invention also provides a transformant introduced with a gene encoding the Fab' fragment of the anti-human NGF antibody of the present invention or a gene encoding the heavy chain variable region and/or the variable region of the light chain of the Fab' fragment of the anti-human NGF antibody of the present invention . Such transformants can be produced, for example, by transforming host cells with the expression vector of the present invention. The host cell used for the preparation of the transformant is not particularly limited as long as it is suitable for the aforementioned expression vector and can be transformed, and various examples include naturally occurring cells or artificially established cells commonly used in the technical field of the present invention. species of cells (for example, bacteria (Escherichia bacteria, Bacillus bacteria), yeast (Saccharomyces, Pichia, etc.), animal cells or insect cells (eg, Sf9), etc.). The conversion can be performed by a method known per se.

Preferably, the transformant of the present invention is a polynucleotide comprising a sequence encoding the heavy chain variable region of the Fab' fragment of the present invention and a sequence comprising the sequence encoding the light chain variable region of the Fab' fragment of the present invention The host cell transformed with the expression vector of the polynucleotide, or an expression vector containing a polynucleotide comprising a sequence encoding the heavy chain variable region of the Fab' fragment of the present invention and a polynucleotide comprising the sequence encoding the Fab' fragment of the present invention The host cell after transformation with the expression vector of the polynucleotide of the sequence of the light chain variable region. More preferably, the transformant of the present invention is expressed using a polynucleotide comprising a sequence encoding the heavy chain fragment of the aforementioned Fab' fragment of the present invention and a polynucleotide comprising a sequence encoding the light chain of the Fab' fragment A host cell transformed with a vector, or an expression vector comprising a polynucleotide comprising a sequence encoding a heavy chain fragment of the Fab' fragment of the present invention and a polynucleotide comprising a sequence encoding a light chain of the Fab' fragment of the present invention host cells transformed with the expression vector.

The present invention also provides the production method of the anti-human NGF antibody Fab' fragment of the present invention, comprising making the gene encoding the anti-human NGF antibody Fab' fragment of the present invention or the heavy chain encoding the anti-human NGF antibody Fab' fragment of the present invention variable The step of expressing the gene of the light chain variable region and/or the light chain variable region in the host cell includes the step of using the above-mentioned transformant. Preferably, the host cell used in the method is a host cell transformed with the aforementioned expression vector of the present invention, and may contain polynucleoside sequences encoding the heavy chain variable region of the Fab' fragment of the present invention, respectively or at the same time. acid and a polynucleotide comprising a sequence encoding the light chain variable region of the Fab' fragment.

In the production of the anti-human NGF antibody Fab' fragment of the present invention, the transformant can be cultured in a nutrient medium. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source required for the growth of the transformant. As a carbon source, for example, glucose, dextran, soluble starch, sucrose, etc., as an inorganic nitrogen source or an organic nitrogen source, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat paste, soybean meal, potato extract, etc. In addition, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.) )wait).

The transformant is cultivated by a method known per se. Culture conditions such as temperature, pH of the medium, and culture time can be appropriately selected. For example, when the host is an animal cell, as a medium, MEM medium (Science, Vol.122, p.501, 1952), DMEM medium (Virology , Vol.8, p.396, 1959), RPMI1640 medium (J.Am.Med.Assoc., Vol.199, p.519, 1967), 199 medium (proc.Soc.Exp.Biol.Med. , Vol.73, p.1, 1950) and so on. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40° C. for about 15 to 72 hours, and aeration and stirring may be performed as necessary. When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. 8. Cultivation is usually performed at about 20 to 40°C for 15 to 100 hours, and aeration and stirring may be performed as necessary. When the host is bacteria, actinomycetes, yeast, or filamentous fungi, for example, a liquid medium containing the above-mentioned nutrient sources is suitable. A medium with a pH of 5-8 is preferred. When the host is Escherichia coli, as a preferable medium, LB medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972) and the like can be exemplified. In this case, the culture can be performed usually at 14 to 43° C. for about 3 to 24 hours, and aeration and stirring can be performed at the same time as necessary. When the host is bacteria belonging to the genus Bacillus, it can usually be carried out at 30 to 40° C. for about 16 to 96 hours, and aeration and stirring can be performed at the same time as necessary. When the host is yeast, examples of the medium include Burkholder's minimal medium (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980), and the pH is preferably 5-8. Cultivation is usually carried out at about 20 to 35° C. for about 14 to 144 hours, and aeration and stirring may be performed as necessary.

The anti-human NGF antibody Fab' fragment of the present invention can be recovered, preferably isolated and purified from the above-mentioned transformant by culturing the transformant. As separation and purification methods, for example, methods using solubility such as salting out and solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Ion exchange chromatography, hydroxyapatite chromatography, etc. using charged methods, affinity chromatography, etc. using specific affinity methods, reversed-phase high-performance liquid chromatography, etc., using hydrophobicity differences, isoelectric focusing, etc. The method of isoelectric point difference, etc.

The present invention has been fully described above, and specific examples to be referred to for deepening understanding are provided here, but these examples are for the purpose of illustration and do not limit the present invention.

Example

Regarding the part using a commercially available kit, reagent, etc., unless otherwise specified, the experiment was performed in accordance with the accompanying protocol.

(Example 1: Immunization to VelocImmune mice)

Antibodies to human NGF were obtained by immunizing VelocImmune mice. In order to increase the diversity of the obtained antibodies, the present inventors conducted research on various immunization methods, administration routes, adjuvants, immunization time, and the like. Using human βNGF (R&D System Co., Ltd.) as an immunogen, the method of dissolving human βNGF and mixing it with an adjuvant for immunization, and heat denaturation of human βNGF (under 0.5% SDS solution, 80°C, 10 minutes) and The method of immunization with adjuvant mixture was studied. As the route of administration, plantar administration and intraperitoneal administration were investigated. As adjuvants, TiterMax Gold (CytRx), complete Freund's adjuvant (Sigma), incomplete Freund's adjuvant (Sigma), and RIBI adjuvant (Corixa) were investigated. In addition, as further added immune activators, CpG oligonucleotides and aluminum phosphate gel (BRENNTAG) were investigated. As the immunization time, 3 weeks to 14 weeks were studied. After several immunizations, blood was collected from the tail vein of the mice, and the titer was monitored to select VelocImmune mice that produced antibodies that bind to human NGF.

The titer determination was performed using the following standard ELISA method. 10 ng of human βNGF was added to each well of a Maxisorp384 plate (Nunc), and incubated overnight at 4° C. to immobilize it. The next day, after washing the plate once with washing solution (TBST: tris buffer solution (TBS) containing 0.05% Tween-20), add blocking agent (TBST containing 20% BlockingOne (Nacalai Tesque)), and Let stand at room temperature for 1 hour. After washing once with TBST washing solution, a dilution series of the collected blood was prepared and added. After incubating at room temperature for 1 hour, wash 3 times with TBST washing solution, add horseradish peroxidase-labeled rabbit anti-mouse Ig antibody (HRP-goatanti- mouseIgantibody; Zymed Corporation). After incubation for 1 hour at room temperature, wash 3 times with TBST washing solution. After adding TMB chromogenic reagent (SumitomoBakelite Company) and standing at room temperature for 10 minutes, a stop solution (2 mol/L sulfuric acid) was added to stop the reaction, and the absorbance at 450 nm was measured.

(Example 2: Production of hybridomas producing anti-human NGF antibody)

Final immunization (intravenous or intraperitoneal administration of the antigen) was performed on the mice selected after confirming the increase in the antibody titer. Lymphocytes were collected by removing the spleen, lymph nodes, etc. of the immunized mice according to a conventional method, and they were fused with mouse myeloma cells SP2/0 to prepare hybridomas. The hybridoma was limitedly diluted and monoclonalized, and on this basis, the antibody was purified from the supernatant using a protein A or protein G column (GE HEALTHCARE).

(Example 3: Evaluation of NGF-trkA binding inhibition)

Human βNGF (R&D System Company) was reacted with EZ-LINK5-(biotinamide) pentylamine (PIERCE Company) at room temperature and in the dark for 30 minutes to carry out biotin labeling, and the excess biotin was removed with a desalting column to obtain biotin Tagged human βNGF. In addition, in the following Examples 6 and 7, it was confirmed that the biological activity of the produced biotin-labeled human βNGF was equivalent to that of native human βNGF.

Inhibitory activity was measured using the following method. 60 ng of human trkA (R&D Systems) was added to each well of a white Maxisorp384 plate (Nunc), and incubated overnight at 4°C to immobilize it. The next day, after the plate was washed once with TBST washing solution, a blocking agent (TBST containing 20% Blocking One (Nacalai Tesque)) was added, and allowed to stand at room temperature for 1 hour. Next, the biotin-labeled human βNGF (0.2 μg/ml) prepared above was mixed with the antibody obtained in Example 2, and the resultant was added to the blocked trkA solid-phase plate. After incubating at room temperature for 1 hour, they were washed 3 times with TBST washing solution, and alkaline phosphatase-labeled streptavidin (PIERCE Company) was added. After incubating at room temperature for 1 hour, they were washed three times with TBST washing solution, and APU4 (BioFX) was added as a chemiluminescent detection reagent, and the chemiluminescent amount was measured with an EnVision counter (PerkinElmer).

(Example 4: Species cross-reactivity evaluation)

When the antibody has cross-reactivity to mouse βNGF, the antibody can be used to evaluate drug efficacy in a mouse pathological model. Therefore, in the method of Example 3, mouse βNGF (R&D Systems) was used to prepare biotin-labeled mouse βNGF, and the cross-reactivity of the antibody to mouse βNGF was evaluated.

(Example 5: Evaluation of Binding Specificity)

The binding specificity of the antibody to NGF was evaluated using the ELISA method described in Example 1. Specifically, using NT-3, the family molecule with the highest homology to NGF, in the method of Example 1, 20 ng of human NT-3 (PeproTech) was added to each well and carried out on a plate for solid phase After transformation, evaluate.

(Example 6: NGF-trkA signal inhibition evaluation)

The inhibitory activity of the antibodies against NGF-trkA signaling was evaluated. NGF causes an increase in intracellular calcium (Ca2+) concentration through trkA as its receptor. Usually, this change in Ca2+ concentration can be evaluated using the intracellular Ca2+ concentration measurement system (FLIPR; Molecular Devices, Inc.) in the presence of a calcium indicator reagent.

Inhibitory activity was measured using the following method. On the day before the experiment, HEK293 cells (WO2009/054468) stably expressing human trkA were injected into 96-well poly-D-lysine-coated plates (Becton Dickinson, Japan) at a rate of ² ×104 cells/well. , cultured overnight. The next day, the medium was replaced with DMEM medium (containing 3.6 mM sodium hydroxide (NaOH), 2.5 mM probenecid (Sigma Company)) containing a calcium indicator reagent (Fluo4-AM; Tongrentang Company) and incubated at 37 °C. Let stand for 30 minutes. Next, wash solution (Hanks'Balanced salt solution (HBSS) (20mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3.6mM sodium hydroxide, 2.5mM propane Sulfur (Sigma Company), 0.1% bovine serum albumin) washed the cells twice, and replaced the medium with the washing solution at 150 μl/well. The cell plate was set in FLIPR. Through the operation of FLIPR, 50 μl/well Add the mixed solution of the antibody and NGF obtained in Example 2 (NGF final concentration 100ng/ml) to measure the change of intracellular Ca ²⁺ concentration. Calculate the difference between the maximum value and the minimum value of the intracellular Ca ²⁺ concentration change , and save it as measurement data.

(Example 7: Evaluation of NGF-dependent cell survival signal inhibition)

In the case of PC12 cells naturally expressing trkA and p75 receptors cultured under serum-free conditions, NGF can maintain the survival of cells for several days. The inhibitory activity of the antibodies against NGF-dependent cell survival signals was evaluated using the following method.

PC12 cells were seeded on 96-well collagen-coated plates (ASAHI TECHNO) at ¹ × 104 cells per well, and were treated with 2.5% fetal bovine serum and 15% inactivated horse serum (Invitrogen). F12K medium (Invitrogen) was incubated overnight at 37°C, 5% CO ₂ . The next day, the medium was changed to serum-free conditions for F12K only. After 1 hour, antibodies and human βNGF (final concentration 50ng/ml) were added and cultured for 72 hours. Then, the culture medium was removed with a suction device, and the viability of the cells was measured by the endogenous ATP quantification reagent of the cells (CellTiter Glo; Promega Company).

(Example 8: Production of Fab fragments)

Using a Fab preparation kit (Pierce), the digestive enzyme papain-conjugated gel was added to antibody 1 mg/ml, and treated at 37°C for 3 hours. The treated reaction solution was added to a protein G column (GE HEALTHCARE), and the cleaved Fc and unreacted IgG were removed by adsorption on the column, and the eluted fraction was recovered to obtain a Fab fragment. The obtained Fab fragments were evaluated by the tests described in Example 3, Example 6, and Example 7.

From the evaluation results of Examples 3 to 8, it can be confirmed that the antibodies (chimeric antibodies) named 1-15 have high neutralizing activity, species cross-reactivity, and binding specificity, and even when they become monovalent antibody fragments It can also maintain high neutralizing activity even under the condition of

(Example 9: Determination of antibody gene sequence)

For the identified 1-15 antibodies, the inventors cloned the genes encoding the heavy and light chains of the antibodies from hybridomas. Specifically, 1×10 ⁵ or more hybridoma clones were prepared, suspended in the RLT buffer contained in the RNeasy Mini Kit (QIAGEN), and then disrupted using a QIAshredder (QIAGEN). Then, RNA was extracted according to the experimental protocol, and the extracted RNA was used as a template to synthesize cDNA using a DNA amplification kit (SMARTerRACE cDNA Amplification kit; Clontech Company). Using the obtained cDNA, a PCR reaction was performed to extend and amplify the variable regions of the heavy chain and the light chain. The PCR product was sequenced by a direct sequencer (ABI PRISM3100; Applied Biosystems). Then, the PCR product was recombined with pCR3.1-TOPO (Invitrogen Company) and other PCR product subcloning vectors, and then the gene sequence was analyzed for sequence determination.

The nucleotide sequence of the heavy chain variable region of the determined 1-15 antibody is represented by SEQ ID NO: 1, the amino acid sequence is represented by SEQ ID NO: 2, and the nucleotide sequence of the light chain variable region of the antibody is represented by SEQ ID NO: 3 , and the amino acid sequence is represented by SEQ ID NO: 4. The CDR1, CDR2, and CDR3 of the heavy chain variable region of the 1-15 antibody are the 31st to 35th, 50th to 65th, and 95th to 102nd regions of the heavy chain variable region based on Kabat numbering, respectively. The 31st-35th, 50th-65th, and 98th-110th amino acid sequences of SEQ ID NO: 2 constitute. The CDR1, CDR2, and CDR3 of the light chain variable region of the 1-15 antibody are the 24th to 34th, 50th to 56th, and 89th to 97th regions of the light chain variable region based on Kabat numbering, respectively. The amino acid sequence of the 24th to 39th, the 55th to 61st and the 94th to 102nd of SEQ ID NO: 4 constitutes.

(Example 10: Production of Mutants of Sugar Chain Modification Sites in Variable Regions)

The heavy chain variable region amino acids (SEQ ID NO: 2) of the aforementioned 1-15 antibodies contain an N-type sugar chain modification motif sequence of N-X-(T/S). Specifically, Asn (N52) at position 52 based on Kabat numbering in the heavy chain variable region shown in SEQ ID NO: 2 corresponds to a sugar chain modification site. When a sugar chain modification site exists, sugar chains are added to the antibody during cell culture, and it is known that sugar chain addition depends on culture conditions and the host expressing it. That is, even in the same established antibody-producing cells, the degree of addition of sugar chains may vary depending on the culture conditions (medium, cell density, etc.), and it may be difficult to obtain antibody drugs of uniform quality. Therefore, the present inventors produced 1-15 (N52D) in which a mutation was introduced into N52 in the heavy chain variable region of the 1-15 antibody.

The nucleotide sequence of the heavy chain variable region of 1-15 (N52D) produced is represented by SEQ ID NO: 5, and the amino acid sequence is represented by SEQ ID NO: 6. CDR1, CDR2, and CDR3 of the heavy chain variable region of the 1-15 (N52D) antibody are the 31st to 35th, 50th to 65th, and 95th to 102nd regions of the heavy chain variable region based on Kabat numbering, respectively , consisting of the 31st to 35th, 50th to 65th, and 98th to 110th amino acid sequences of SEQ ID NO: 6.

(Example 11: Production of fully human antibody Fab' fragment)

Each fully human antibody Fab' fragment was prepared using the aforementioned 1-15 and 1-15 (N52D) heavy chain variable regions and 1-15 light chain variable regions.

A signal sequence was connected to the 5' side of each heavy chain variable region gene of 1-15 and 1-15 (N52D), and then the constant region gene of human Igγ1 was connected to the 3' side (Man Sung Co et al., (1992) JImmunol. Vol.148(4):1149-1154), the heavy chain fragment gene was inserted into the GS vector pEE6.4 (Lonza Biologics). Here, in order to express as a Fab' fragment, in the heavy chain constant region gene, Cys at position 226 based on the EU index (corresponding to Cys at position 230 in the amino acid sequences of SEQ ID NOs. 8 and 10 described later) A stop codon was inserted after the codon. In addition, the signal sequence was connected to the 5' side of the light chain variable region gene of 1-15, and then the constant region gene of human κ chain (ManSungCo et al., above) was connected to the 3' side, and the light chain gene was inserted into the GS vector pEE12 .4 (Lonza Biologics).

The Fab' fragments were expressed by two methods of transient expression and stable expression. In the transient expression, for FreeStyle293 cells (Invitrogen) cultured to about 1 million cells/mL in FreeStyle293 expression medium (Invitrogen), use 293 フェクチン (Invitrogen) for the aforementioned heavy chain fragment and light chain GS vector Transfection was performed and cultured for 7 days. In stable expression, the aforementioned two GS vectors were cut with NotI and PvuI restriction enzymes, connected using a DNA ligation kit (Bao Biological Co., Ltd.), and a GS vector with two genes inserted into the heavy chain fragment and the light chain was constructed. . The expression vector encodes heavy chain fragments, light chains and glutamine synthetase (Glutamine synthetase), and is expressed by transfecting CHO-K1SV cells. After expression by each method, the culture supernatant was purified using KappaSelect (GE HEALTHCARE) to obtain each Fab' fragment.

The nucleotide sequence of the heavy chain fragment of the produced fully human 1-15 antibody Fab' fragment (also referred to as 1-15-Fab') is represented by SEQ ID NO: 7, and the amino acid sequence is represented by SEQ ID NO: 8.

The nucleotide sequence of the heavy chain fragment of the produced fully human 1-15(N52D) antibody Fab' fragment (also called 1-15(N52D)-Fab') is represented by SEQ ID NO: 9, and the amino acid sequence is represented by SEQ ID NO: 10 means.

The light chains of the respective Fab' fragments are identical, and their base sequence is represented by SEQ ID NO: 11, and their amino acid sequence is represented by SEQ ID NO: 12.

(Example 12: Evaluation of neutralizing activity and expression level of fully human antibody Fab' fragments)

1-15-Fab' and 1-15(N52D)-Fab' obtained in Example 11 were evaluated by the tests described in Examples 3 and 6. In the test of Example 3, the IC50 of 1-15-Fab' and 1-15(N52D)-Fab' were 0.17 µg/ml and 0.18 µg/ml, respectively. In the test of Example 6, the IC50 of 1-15-Fab' and 1-15(N52D)-Fab' were 0.021 µg/ml and 0.018 µg/ml, respectively. From these results, it was confirmed that 1-15(N52D)-Fab' maintained the same level of neutralizing activity as that of 1-15-Fab' before the modification, and that the neutralizing activity was not affected even if the mutation was introduced.

In addition, each Fab' fragment was stably expressed, and the amount of antibody production in the culture supernatant of the stably expressing cell pool was measured. As a result, the concentrations in the culture supernatants of 1-15-Fab' and 1-15(N52D)-Fab' were 86 mg/L and 106 mg/L, respectively, showing that 1-15(N52D)-Fab' was Changes to higher amounts of 1-15-Fab' produced antibodies.

(Example 13: Production of PEGylated Fab' fragments and evaluation of neutralizing activity)

Next, the present inventors introduced PEG into the aforementioned 1-15(N52D)-Fab'. After purification with KappaSelect, the Fab' fragment was reduced with TCEP hydrochloride (Tris(2-carboxyethyl)phosphine HCl, tris(2-carbonylethyl)phosphine hydrochloride) to obtain a structure capable of PEGylation .

Specifically, TCEP was added so as to reach 1 mM to a Fab' fragment solution adjusted to 1.2 mg/ml with 20 mM sodium phosphate buffer (pH 6.8), reacted at 37 °C for 2 hours, and then buffered with 20 mM sodium acetate solution (pH5.0) to adjust the pH. The resultant was adsorbed on a cation exchange resin (SP-5PW; manufactured by Tosoh), and eluted with a NaCl gradient to recover the main peak. The obtained Fab' fragments were diluted to 1 mg/ml with 20 mM sodium phosphate buffer (pH 6.8), adjusted to pH 6.8, and allowed to stand overnight at 4°C for natural oxidation. 40 kDa PEG (SUNBRIGHTGL2-400MA; NOF Corporation) was added thereto to a final concentration of 0.1 mM, and after standing at room temperature for 2 hours, it was left at 4°C overnight. This PEG has a maleimide group at its terminal, and therefore reacts rapidly with Cys (C226 based on EU index; Cys at position 230 of SEQ ID NO: 10) at the carboxy-terminal of the heavy chain fragment. The resultant was diluted with 20 mM sodium acetate buffer (pH 4.5), adjusted the pH, and again adsorbed on a cation exchange resin (SP-5PW; manufactured by Tosoh), and eluted with a gradient of NaCl to recover the main peak. By this operation, the PEGylated Fab' fragment was purified. This PEGylated 1-15(N52D)-Fab' is also referred to as 1-15(N52D)-Fab'-PEG.

The neutralizing activity of 1-15(N52D)-Fab' before and after PEGylation was evaluated by the method shown in Example 3. As a result, the IC50 of 1-15(N52D)-Fab' was 0.15 μg/ml, while the IC50 of 1-15(N52D)-Fab'-PEG was 0.12 μg/ml (in terms of Fab' fragment concentration), It was confirmed that the neutralization activity of 1-15(N52D)-Fab' was not affected even when PEG was added.

In addition, using the method of Example 6, the neutralizing activities of 1-15(N52D)-Fab'-PEG and the existing anti-human NGF antibody Tanezumab on human and mouse NGF were compared. As a result, the IC50 of 1-15(N52D)-Fab'-PEG against human NGF was 0.051 μg/ml, and that of mouse NGF was 0.069 μg/ml. In contrast, the IC50 of Tanezumab against human NGF was 0.17 μg/ml, With respect to mouse NGF at 0.23 μg/ml, it was confirmed that 1-15(N52D)-Fab'-PEG has a neutralizing activity about 3.3 times stronger than that of Tanezumab against both human and mouse NGF.

(Example 14: Analgesic effect test using mouse adjuvant-induced arthritis model)

The present inventors evaluated the analgesic effect of the aforementioned 1-15(N52D)-Fab'-PEG on a mouse adjuvant-induced arthritis model.

Intravenous administration of 1-15(N52D)-Fab'-PEG (0.03mg/kg, 0.1mg/kg, 0.3mg/kg, administration volume 10mL/kg) in mice, 25μL on the sole of the hind limb Complete Freund's adjuvant (Sigma Company) 1mg/mL caused pain. Standing activity was measured for 20 minutes within 24 hours of induction. Specifically, using the SuperMex Spontaneous Movement Measurement System (Muromachi Machinery), the number of times mice spontaneously stood up for 20 minutes was automatically measured with an infrared beam sensor (Matson et al., JPET320: 194-201, 2007). As a comparative control, the existing antibody Tanezumab was used. As a result, the analgesic effect of intravenous administration of Tanezumab was ED50 = 0.27 mg/kg, whereas 1-15(N52D)-Fab'-PEG exhibited an analgesic effect with ED50 = 0.11 mg/kg, showing that Out about 3 times the effectiveness.

(Example 15: Rat placenta passage test)

On the 17th day of pregnancy, female rats were intravenously administered 1-15(N52D)-Fab'-PEG or Tanezumab (100mg/kg, dosage volume 10mL/kg), and the antibodies in the maternal and fetal blood were determined 3 days later concentration.

The antibody concentration was measured in the following manner. 25 ng of human βNGF (R&D Systems) was added to each well of a multi-array plate (standard) 96 plate (MesoScale Discovery), and left to stand at room temperature for 1 hour to perform immobilization. After the plate was washed three times with TBST washing solution, a blocking agent (1% casein TBS; Thermofisher Co.) was added and left to stand at room temperature for 1 hour. Next, blood samples obtained by diluting time-collected blood were added to the blocked human βNGF solid-phase plate. After reacting with stirring at room temperature for 60 minutes, the cells were washed three times with TBST washing solution, and a biotin-labeled anti-human kappa antibody (Institute of Immunobiology) was added. After reacting for 60 minutes while stirring at room temperature, they were washed three times with TBST washing solution, and SULFO-TAG-labeled streptavidin (Meso Scale Discovery Company) was added. After reacting for 60 minutes while stirring at room temperature, wash with TBST washing solution 3 times, add Read BufferT (Meso Scale Discovery Company), and measure the amount of electrochemiluminescence with SECTOR Imager6000 (Meso Scale Discovery Company).

As a result of this experiment in 3 mother rats, the average antibody concentrations of 1-15(N52D)-Fab’-PEG and Tanezumab in blood of rat mothers 3 days later were 12.1 μg/ml and 7.1 μg/ml, respectively. On the other hand, regarding the fetal blood antibody concentrations of 3 fetuses (9 cases in total) taken from each rat mother, the blood concentration of 1-15(N52D)-Fab'-PEG was undetectable in all cases. In contrast, the blood concentration of Tanezumab was 5.39 μg/ml on average. That is, the fetal mobility of Tanezumab was 75.9%, whereas the fetal mobility of 1-15(N52D)-Fab'-PEG was 0.08% or less of the detection limit. It can be seen from this that 1-15(N52D)-Fab'-PEG can avoid the risk of side effects caused by NGF inhibition in the fetus, and is a drug with excellent safety.

(Example 16: Formation of Immune Complex (IC))

Whether or not 1-15(N52D)-Fab'-PEG forms an IC, and to what extent the size of the formed IC is evaluated. Specifically, 1-15(N52D)-Fab'-PEG was mixed with 1 mg/ml human βNGF (R&D Systems) at a molar ratio of 1:1, and incubated at room temperature for 3 hours to form IC. The particle size and distribution of IC in this reaction solution were measured using Zetasizer Nano (Malvern Co.), a device for measuring dynamic light scattering. Zetasizar v6.01 (Malvern Co.) was used for the analysis, and the particle size was expressed as a value (d.nm) after analysis by intensity (%).

The measured particle sizes are shown in Table 1 below. In this experiment, the average particle size of NGF alone was 6.2 nm. Tanezumab alone appeared peak size at 11.7nm. After incubation of Tanezumab and NGF to form IC, the peak size was shifted to 91.3nm. On the other hand, when an antibody that does not bind to NGF was used as a control antibody, the peak size was still 11.7 nm. Considering the displacement width, Tanezumab and NGF each form a giant molecule in which multiple molecules are bound, and it is presumed that an IC of a huge size is formed. On the other hand, as a result of measuring the IC formation between 1-15(N52D)-Fab'-PEG and NGF, the peak size shifted from 18.1 nm to 24.4 nm. Considering the shift width, reflecting only 1:1 binding, it was shown that no lattice was formed in 1-15(N52D)-Fab’-PEG.

Table 1

(Example 17: Lesion tissue migration)

Subcutaneous administration of collagen (type 2 collagen derived from bovine joints, 10 mg/mL; Collagen Technology Workshop) and complete Freund's adjuvant (0.5 mg/mL; DIFCO) was subcutaneously administered to the foot joints of male DBA/1 mice 1:1 emulsion to make collagen-induced arthritis model. The lotion was reapplied 4 weeks after induction to allow the onset of arthritis. The incidence of hindlimb arthritis (score, size of swelling) was observed, and the mice were divided into groups. For the 1 mg/ml PBS solution of 1-15(N52D)-Fab'-PEG and Tanezumab, SAIVI™ Rapid Antibody Labeling Kit, AlexaFluor (registered trademark) 680 (Life Technologies) was used for fluorescent labeling. These were administered from the tail vein at 2 mg/kg, respectively (N=4). Using IVIS Spectrum (Caliper/Xenogen), the fluorescence accumulated in the swollen sole was analyzed from 1 hour to 50 hours after administration, and the fluorescence intensity was quantified.

Fig. 1 shows the temporal change of the antibody retention amount in the plantar. Compared with Tanezumab, 1-15(N52D)-Fab'-PEG showed a significant lesion tissue retention effect, and the effect lasted up to 48 hours. From these results, it can be considered that 1-15(N52D)-Fab'-PEG can effectively exert an analgesic effect, an analgesic effect can be expected at a low dose above the drug potency, and can be selectively accumulated in the lesion site, so it can be It is expected to become a drug with excellent safety.

(Example 18: Preparation of amino acid addition body of Fab' fragment)

In order to improve the introduction efficiency of PEG, the inventors made 1-15(N52D)-Fab' by adding 2 alanine (A) or proline ( P) Fab' fragments were expressed and purified. In the preparation of these Fab' fragments, the same method as in Example 11 was used, here, two alanines were inserted after the codon of the carboxy-terminal Cys residue of the heavy chain fragment of 1-15(N52D)-Fab' acid or proline codon, followed by a stop codon.

The heavy chain of 1-15(N52D-A)-Fab' (full human 1-15(N52D-A) antibody Fab' fragment; also known as 1-15(N52D-A)-Fab') with the addition of alanine The base sequence of the fragment is represented by SEQ ID NO: 13, and the amino acid sequence is represented by SEQ ID NO: 14. The heavy chain of 1-15(N52D-P)-Fab' (full human 1-15(N52D-P) antibody Fab' fragment; also known as 1-15(N52D-P)-Fab') with added proline The base sequence of the fragment is represented by SEQ ID NO: 15, and the amino acid sequence is represented by SEQ ID NO: 16. It should be noted that the light chain of each Fab' fragment is identical to the light chain of 1-15(N52D)-Fab', its base sequence is represented by SEQ ID NO: 11, and its amino acid sequence is represented by SEQ ID NO: 12.

(Example 19: Preparation of PEGylated 1-15(N52D-A)-Fab' and neutralizing activity and pharmacological evaluation)

For 1-15(N52D-A)-Fab', PEGylated 1-15(N52D-A)-Fab' (hereinafter also referred to as 1-15(N52D- A)-Fab'-PEG).

For 1-15(N52D-A)-Fab'-PEG, the neutralizing activity was evaluated using the method shown in Example 3. As a result, the IC50 of 1-15(N52D)-Fab'-PEG was 0.081±0.034μg/ml, while the IC50 of 1-15(N52D-A)-Fab'-PEG was 0.074±0.021μg/ml ml. In addition, at this time, the IC50 of Tanezumab was 0.410±0.099 μg/ml.

Next, the neutralization activity was compared using the method shown in Example 6. As a result, the IC50 of 1-15(N52D)-Fab'-PEG against human NGF was 0.061±0.011 μg/ml, while the IC50 of 1-15(N52D-A)-Fab'-PEG was 0.064± 0.028 μg/ml.

In addition, the analgesic effect in the adjuvant-induced arthritis model was evaluated using the method shown in Example 14. As a result, 1-15(N52D)-Fab'-PEG exerted analgesic effect at ED50=0.14 mg/kg, while 1-15(N52D-A)-Fab'-PEG exhibited analgesic effect at ED50=0.21 mg/kg kg exert analgesic effect.

From the above, it was confirmed that even if two alanines were added after the Cys residue at the carboxyl terminal, the neutralizing activity and pharmacological activity were not affected.

(Example 20: Evaluation of the binding affinity of 1-15(N52D-A)-Fab'-PEG)

The binding thermodynamics of 1-15(N52D-A)-Fab'-PEG and Tanezumab to NGF antigen were studied by isothermal titration calorimetry (Isothermal titration calorimetry; ITC) (Scappaticci FA, J Natl Cancer Inst. 2007, 99: 1232 -9. Velazquez-Campoy, A., et al, CurrProtoc Cell Biol. 2004, Chapter 17, Unit 17-18.). All measurements were performed using Auto-iTC200 manufactured by GE Healthcare. In the experiment, in order to evaluate the binding between the monovalent Fab' fragment and one molecule of the antigen, the following concentrations were tested, and all the tests were performed in PBS solvent. Specifically, 1.4 μL of human βNGF 44 μM (R&D systems) contained in a syringe for titration was titrated 30 times to the antibody-filled sample (3 μM of 1-15(N52D-A)-Fab'-PEG or 1.5 μM Tanezumab) in the calorimeter pool to detect the heat generated at this time. Using the software attached to the device, the data obtained are analyzed by the Single site binding model, thereby estimating the binding affinity (Kd), binding ratio (n), binding free energy (ΔG), and binding enthalpy (ΔH) associated with antigen-antibody binding , and binding entropy (-TΔS). The results are shown in Table 2.

As a result, the Kd value of Tanezumab was 20.41nM, whereas the Kd value of 1-15(N52D-A)-Fab'-PEG was 1.49nM, and that of 1-15(N52D-A)-Fab'-PEG The binding affinity is more than 10 times stronger than Tanezumab.

Table 2

Kd(nM)ΔG (cal/mil) H(cal/mol) -TS(cal/mo) Tanezumab 20.41 -10490 -4759 -5731 1-15(N52D-A)Fab'-PEG 1.49 -12041 -20806 8765

(Example 21: Preparation of PEGylated 1-15(N52D-A)-Fab' with various PEG sizes and evaluation of neutralizing activity)

For 1-15(N52D-A)-Fab' prepared in Example 18, 5kDa PEG or 10kDa PEG was combined using the same procedure as in Example 13. Specifically, after reducing the Fab' fragment solution prepared with 20 mM tris hydrochloric acid buffer (pH 7.4) with TCEP, the Fab' fragment was recovered using a desalting column. PEG (SUNBRIGHTGL2-50MA or SUNBRIGHT GL2-100MA; both from NOF) was added to the obtained Fab' fragments, and left to stand overnight at 4°C. The resulting 1-15(N52D-A)-Fab' combined with 5kDaPEG or 10kDaPEG is called 1-15(N52D-A)-Fab'-5kPEG and 1-15(N52D-A)-Fab'-10kPEG respectively .

Next, using the method shown in Example 6, the neutralizing activity of each PEGylated Fab' fragment was compared. As a comparative control, 1-15(N52D-A)-Fab'-PEG (conjugated 40 kDa PEG; hereinafter also referred to as 1-15(N52D-A)-Fab'-40kPEG) produced in Example 19 was used. In this experiment, the final concentration of NGF was 50ng/ml. As a result, the IC50 of 1-15(N52D-A)-Fab'-5kPEG, 1-15(N52D-A)-Fab'-10kPEG, and 1-15(N52D-A)-Fab'-40kPEG were 0.030 μg/ml, 0.028 μg/ml, and 0.023 μg/ml. From this result, it can be seen that the neutralization activity of the Fab' fragment is not affected by the size of PEG between 5kDa and 40kDa.

(Example 22: Mouse PK evaluation of PEGylated 1-15(N52D-A)-Fab' with various PEG sizes)

Mouse PK evaluation of various PEGylated 1-15(N52D-A)-Fab' was performed. Specifically, after intravenous administration of 0.3 mg/kg of various PEGylated 1-15(N52D-A)-Fab', 1, 4, 8, 12, 24, 48, 72, 96, and 168 hours respectively for blood collection. The amount of the antibody to be tested in the obtained blood was measured by the sandwich ELISA method. Specifically, the antibody to be tested was added to an NGF-immobilized MSD plate (manufactured by Meso Scale Discovery). Anti-human Kappa antibody labeled with biotin recognizes the test antibody bound to the plate, and it is detected with SULFO-TAG labeled streptavidin. The blood concentration was calculated by preparing a calibration curve using each standard product. Calculate the blood half-life (T1/2: hours) from the calculated blood concentration. As a result, T1/2 13.8±2.2 hours, 17.7±0.4 hours, and 39.2±3.7 hours, respectively.

(Example 23: Analgesic effect test using a rat arch incision model)

1-15 (N52D-A)-Fab was evaluated using a rat post-antitomy pain model (Brennan et al. Current Protocols in Pharmacology 2004; 5.34.1-5.34.8) thought to mirror postoperative pain in the clinic Analgesic effect of '-5kPEG and 1-15(N52D-A)-Fab'-10kPEG on postoperative pain.

Specifically, 8 rats in each group were intravenously administered 1-15(N52D-A)-Fab'-5kPEG or -10kPEG (0.1mg/kg, 0.3mg/kg, 1mg/kg, administration volume 1 mL/kg), the arch of the right hind limb was incised 10 mm in a straight line starting from the part 5 mm from the front of the heel to the toe, and two mattress sutures were immediately performed with nylon thread, which caused pain. Pain thresholds near the surgical site were measured at 5 hours, 1 day, 2 days, 3 days, 4 days, and 5 days of induction. In the measurement, a dynamic plantar anesthesiometer manufactured by Ugo Basile Co., Ltd. was used to measure the pressure indicating an escape action by pressing the sole of the rat's foot. As a comparative control, the existing antibody Tanezumab was used.

As a result, the analgesic effect of intravenous administration of Tanezumab on the first day after surgery was ED50 = 0.26 mg/kg, whereas 1-15(N52D-A)-Fab'-5kPEG or -10kPEG both had ED50 = 0.15 mg/kg, exhibited an analgesic effect, showing about 2-fold effectiveness. In addition, significant analgesic effects of 1-15(N52D-A)-Fab'-5kPEG or -10kPEG could be observed up to 3 or 4 days after surgery, respectively.

(Example 24: Evaluation of aggregation stability)

1-15(N52D-A)-Fab'-40kPEG was dissolved to 1mg/ml and 10mg/ml under the conditions of pH5, pH6, pH7.4 and pH9 respectively. These were respectively placed under the condition of 50° C., and the aggregation stability after 2 weeks was evaluated. The aggregation property was evaluated by size exclusion chromatography and measured using 1100 manufactured by Agilent Corporation. The measurement conditions were as follows: 0.1M sodium phosphate (containing 0.2M arginine) (pH 6.8) was used as the mobile phase buffer, and the column used TSKgel Super Sw3000 (TOSOH, 2.0mm ID×300mm). The detection wavelength is 280nm. In the 1 mg/ml test, Tanezumab was used as a comparative antibody, and the results are shown in Table 3. In the 10 mg/ml test, Tanezumab and REGN475 were used as comparative antibodies, and the results are shown in Table 4.

As a result, in Tanezumab and REGN475, a significant increase in the generation of aggregates was observed after 2 weeks, whereas in 1-15(N52D-A)-Fab'-40kPEG, almost no aggregates were observed. From this result, it can be seen that PEGylated 1-15(N52D-A)-Fab' is highly likely to be a drug with excellent storage stability.

table 3

Table 4

- not tested

Industrial availability

The anti-human NGF antibody of the present invention, more specifically, the anti-human NGF antibody Fab' fragment is useful for the prevention or treatment of various diseases in which human NGF is involved in the pathogenesis.

Claims (7)

1. an anti-human NGF antibody Fab ' fragment, it comprises the heavy chain fragment be made up of the aminoacid sequence shown in sequence number 10, sequence number 14 or sequence number 16 and the light chain be made up of the aminoacid sequence shown in sequence number 12.

2. Fab ' fragment as claimed in claim 1, it is combined with polyoxyethylene glycol.

3. an expression vector, it contains: the polynucleotide of the sequence of the polynucleotide comprising the sequence of the heavy chain fragment of Fab ' fragment according to claim 1 of encoding and the light chain that comprises Fab ' fragment according to claim 1 of encoding.

4. a host cell, it is selected from the group be made up of following (a) and (b),

A () is with the host cell transformed containing the polynucleotide of sequence of heavy chain fragment and the expression vector of polynucleotide of sequence of light chain that comprises this Fab ' fragment of coding that comprise Fab ' fragment according to claim 1 of encoding; And

B () is with containing the host cell comprising the expression vector of polynucleotide of sequence of heavy chain fragment of Fab ' fragment according to claim 1 of encoding and the expression vector of polynucleotide of sequence containing the light chain comprising this Fab ' fragment of coding and transform.

5. a method for production anti-human NGF antibody Fab ' fragment, it comprises cultivation host cell according to claim 4 and makes the step of anti-human NGF antibody Fab ' fragment expression.

6. an anti-human NGF antibody Fab ' fragment, it is obtained by method according to claim 5.

7. anti-human NGF antibody Fab ' fragment as claimed in claim 6, it is combined with polyoxyethylene glycol.